Hepatitis C virus (HCV) is the recently recognized causative agent of parenterally transmitted non-A, non-B (NANB) hepatitis. The RNA genome of the virus has been molecularly cloned from the plasma of an experimentally infected chimpanzee (see document number 8 in the list of numbered documents at the end of the specification). Analysis of the sequence of the genome revealed that it is a single-stranded, plus sense RNA of approximately 9500 nucleotides in length. A single long open reading frame of 9033 nucleotides was found, coding for a polyprotein of 3011 amino acid residues. These genomic features and sequence comparisons, combined with knowledge about the size and lability to lipid solvents, indicated that the virus is probably a member of the Flavivirus family (6,22). However, it may be more closely related to the pestivirus genus than classic flaviviruses (27,60).
Based on sequence similarities of both the genome and the predicted polyprotein, as well as on certain features of the polyprotein such as hydrophobicity profiles and acidic/basic amino acid content, it is predicted that proteins of HCV are encoded in the same general regions of the genome as has been determined for the flaviviruses. In the flaviviruses and the related pestiviruses, approximately the amino-terminal one third of the polyprotein constitutes the structural proteins of the virus. These consist of a highly basic nucleocapsid protein termed "C," an envelope-associated glycoprotein termed "M" and a second envelope glycoprotein "E." The non-structural proteins are named NS1 through NS5, but the functions of only NS3 and NS5 have been assigned with certainty. NS3 is the viral protease and probably a helicase, and NS5 is the viral RNA-dependent RNA polymerase.
The HCV polyprotein is processed in a series of proteolytic digestions by a combination of the viral protease and host signalase. The processing of the pestivirus polyprotein is different in detail from that of the flaviviruses, but is similar in its general scheme. The proteins of HCV have been determined largely by analogy with the flavivirus proteins, and, like the pestiviruses, they may also differ in details of size, number, and processing from the flaviviruses. With these caveats, for convenience of nomenclature but not to imply identity in function or processing, hereafter the nonstructural region under study that is analogous to the flavivirus NS5 region RNA polymerase is referred to as the "NS5 region" or "NS5 protein" of HCV.
Diagnostic tests based on expressed viral antigens have been developed and seroepidemiologic surveys using these assays have been performed (33). Studies on the immune response to HCV infections are still at a formative stage. The commercially available antibody assay measures antibody to an HCV antigen expressed in yeast by recombinant DNA technology. This antigen, termed "C100-3", is derived from the non-structural protein coding region of the viral genome and probably represents a portion of NS4 (33). Newer versions of the assay include antigens from NS3 (protease/helicase) and the internal nucleocapsid protein, C (64). None of these antibodies is thought to be protective, but each is commonly found in chronically infected individuals.
HCV is not only the cause of most cases of parenterally acquired non-B hepatitis, but also is responsible for a large portion of sporadic community acquired acute viral hepatitis, chronic hepatitis of unknown origin, as well as cryptogenic cirrhosis and probably hepatocellular carcinoma (2,16,31,50). It is the propensity of this virus to cause chronic infections and chronic liver disease that makes it such a medically important problem. Treatment of chronic HCV liver disease by .alpha.-interferon therapy has recently been approved by the Food and Drug Administration (FDA) in the United States. However, less than half the patients respond, and of the responders approximately 50% relapse after treatment is stopped (16).
Accordingly, there is an important need for a vaccine to protect against infection by this virus. While the present diagnostic tests are based on detection of serum antibody to expressed viral proteins, to date all proteins recognized by such antibodies have represented either non-structural viral proteins or internal components of the virus particle. It is not clear whether effective neutralizing antibodies to HCV are commonly produced by individuals infected with the virus.
Cytotoxic T lymphocytes (CTL) have been found to mediate protection in vivo against certain virus infections (17,47,48). In hepatitis B infection, CTL are thought to be responsible for the pathogenesis of chronic type B hepatitis and to lyse hepatitis B-virus-infected hepatocytes by recognizing the viral antigen expressed on infected cells (39,42). In the case of HCV, there is no information on the pathogenesis of viral infections. The chronicity of HCV infections as well as histopathologic findings indicate that the virus is probably not directly cytopathic (or cytolytic) in hepatocytes. It is possible that the chronic liver disease associated with HCV infections is immune mediated.
These observations provide reason to believe that CTL specific for HCV may be involved causally in the pathogenesis of HCV-related disease, or that cellular immunity is important for protection or recovery from infection. Previous studies have reported that CD8.sup.+ CTL recognize hepatocytes from patients with chronic NANB hepatitis (28). To date, however, no epitope of HCV recognized by T cells has been identified in any HCV protein.
Accordingly, there is a need to identify an epitope of HCV that is recognized by T cells. Peptides comprising such an epitope can be used to determine the role of CTL in HCV infection and pathogenesis. Thus, such peptides may be used to modulate the HCV-specific CTL response in treatment of the hepatitis or other HCV-related disease, by enhancing a protective effect or blocking a pathogenic effect. In addition, such peptides may be used in a vaccine to prevent infection, by stimulating a protective CTL response.
In other viral models, internal proteins are the major targets of the CTL response (3,30,49,62,69). In previous studies by the present inventors and others, the peptides from an internal protein, reverse transcriptase, of HIV as well as envelope glycoprotein and gag and nef proteins were recognized by CTL in the mouse and human (10,14,24,32,43,57,65). Immunization with the envelope protein may not be practical for HCV because the envelope is relatively highly variable in sequence (27). Therefore, CTL clones may distinguish different isolates of HCV, as has been shown in HIV-1 studies (41,58,59).
In contrast to the substantial amino acid sequence variation in the predicted envelope glycoproteins of HCV (designated "E1" and "E2/NS1"), the internal nucleocapsid proteins, C, nonstructural (NS) region 3, NS4, and NS5 proteins all show greater sequence conservation among HCV isolate groups (27). The coding region of the HCV genome that is analogous to NS5 of the flaviviruses by both its location in the genome and its sequence similarities, is believed to represent the viral replicase.